Following the discovery of the anti-tumor activity of cisplatin (Rosenburg et al, 1969) extensive research has been conducted into areas related to the use of platinum complexes for the treatment of cancer. The anti-tumor activity of platinum compounds is believed to result from the loss of the labile chlorine ligand(s) in vivo to form a reactive mono- or di-aqua complex, which is able to form intra- and inter-strand DNA crosslinks in tumor cells. These crosslinks can result in cell death. Cisplatin (cDDP or cis-diamminedichloroplatinum(II) is the most widely used of the platinum compounds approved for use in human subjects, and is indicated for the treatment of solid tumors, including testicular, ovarian, and head and neck, and in combination with other agents in use against squamous cell carcinoma and small cell lung carcinoma (Sur, et al., 1983).
However, there are significant limitations to the use of cisplatin due to its toxicity. Nephrotoxicity and ototoxicity are typically its dose limiting toxicities. Because of this problem, many researchers have made and tested novel small molecule platinum chelates in the hope of finding new compounds in which the therapeutic index (the ratio between the maximum dose that can be tolerated due to toxicity and the dose which provides efficacy) is improved. Changes in platinum chelate structure might also extend the spectrum of tumor types for which platinum therapy could be effective, and/or alter the toxicity profile. As noted above, labile leaving groups are required for tumorcidal activity, but these functional groups can also contribute to the toxicity of the molecule. Research conducted at the Institute for Cancer Research in the U.K. demonstrated that by replacing the chlorine atoms with other leaving groups, compounds could be obtained with lower nephrotoxicity (Harrap, 1985). This work led to the discovery of carboplatin, a cisplatin analog in which the two coordinated chloride ions are replaced by a chelate of 1,1-cyclobutane-dicarboxylic acid. This chelating group is less labile compared with the chlorine atoms of cisplatin. As a result, compared to cisplatin, higher doses of carboplatin are required for a similar tumorcidal effect, but carboplatin has a higher therapeutic index, and the dose limiting toxicity is myelosuppression rather than nephrotoxicity.
Oxaliplatin is another small platinum chelate approved for human use in Europe. This platinum chelate was the result of research investigating the effect of changes in both the non-labile (amine) ligand of cisplatin as well as the labile ligands. In oxaliplatin, the coordinated ammonia ligands are replaced by a trans-1R,2R-diaminocyclohexane (DACH) chelate, while the labile chlorine ligands are replaced by an oxalic acid chelate. It has been shown that oxaliplatin (and other DACH platinum compounds) have a different activity spectrum when compared with cisplatin and carboplatin in the NCI human tumor screen (Paull et al. 1989), and oxaliplatin was subsequently developed for the treatment of colorectal cancer. The dose limiting toxicity of oxaliplatin is sensory neuropathy.
Many other small platinum complexes have been investigated as potential chemotherapeutic agents, but at best, only slight improvements to efficacy and therapeutic index have been achieved. Many of these newer small platinum chelates are inactive or have formulation problems (for example, low solubility in water or poor aqueous stability), and most induce severe toxic side effects including nephrotoxicity, neurotoxicity, myelosuppression, nausea and vomiting. A number of attempts to improve the therapeutic index of the approved platinum complexes have involved either combination therapy, for example, the co-administration of cisplatin and paclitaxel; (Posner et al, 2000) or formulation changes, such as entrapment in liposomes (Steerenberg et al, 1988). There remains a distinct need for new platinum chelates with further improvements in therapeutic index compared with the currently-approved platinum chelates. Such chelates would ideally be water soluble and stable in an aqueous environment, but sufficiently labile in tumor cells to provide species capable of crosslinking DNA and ultimately causing tumor cell death.
Furthermore, improvements to therapeutic index might be achieved by targeting of platinum complexes to tumor cells. Conventional small molecule platinum complexes such as cisplatin, carboplatin, and oxaliplatin are not specifically targeted to tumor cells, and following intravenous administration, they can diffuse into normal cells as readily as they diffuse into tumor cells. Also, their doses are rapidly cleared. At 3 hour post injection 90% of plasma platinum from cisplatin is irreversibly protein bound (Physican""s. Desk Ref. 1997). For cisplatin and carboplatin 25% and 65%, respectively, of the dose is renally secreted within 12 h (DeVita et al. 1993). Improvements in therapeutic index might be possible if platinum complexes are more readily delivered to tumors and/or more readily taken up by tumor cells than normal cells.
One method of tumor targeting which has been extensively reported in the literature involves the labile attachment of a chemotherapeutic compound to a polymer or other macromolecular structure. It has been demonstrated that the concentration of polymers and nanoparticles in tumors exceeds their concentration in normal tissue following intravenous administration (Seymour 1992; Veronese et al. 1999). The mechanism for this preferred tumor accumulation has been termed the xe2x80x9cenhanced permeability and retentionxe2x80x9d (or xe2x80x9cEPRxe2x80x9d) effect (Seymour et al. 1995). Essentially, tumor endothelial cells are more xe2x80x98leakyxe2x80x99 than normal endothelial cells, so polymers and nanoparticles more readily cross the endothelial cell layer in tumors than is the case in normal tissue. Thus, following intravenous administration, polymers and nanoparticles can enter the extracellular fluid of tumor cells much more readily than that of normal cells. Furthermore, lymphatic drainage of the extracellular fluid in tumor cells is much less efficient compared with normal cells. These two factors account for the greater concentration of polymers and nanoparticles in tumors relative to normal tissue relative to small, freely diffusible molecules.
There are already several examples of constructs which provide for the passive targeting of chemotherapeutic agents to tumors through the EPR effect. For example, doxorubicin was attached to a polyhydroxypropylmethacrylamide, (poly(HPMA)), linear polymer backbone via a tetrapeptide designed to be cleaved by lysosomal enzymes. The water-soluble conjugate was termed xe2x80x98PK1xe2x80x99, and has been subject of numerous publications describing its chemistry, pre-clinical testing, and clinical evaluation (for example, Seymour et al, 1990; Pimm et al, 1996; Duncan et al. 1998; Thomson et al. 1999; Minko et al. 2000). Similarly, HPMA was conjugated to paclitaxel and camptothecin for enhanced delivery of these chemotherapeutic molecules to tumors (Fraier et al, 1998; Caiolfa et al. 2000). Both paclitaxel and camptothecin have been attached to other water-soluble polymers for the purpose of improving tumor targeting and drug water solubility (for example, Li et al. 2000 and Conover et al. 1998).
It has been proposed that polymer-platinum conjugates might be used to benefit patients in treating cancer by increasing the solubility of platinum complexes, reducing systemic toxicity, and targeting tumors by the EPR effect (Duncan 1992). Several examples of polymer-platinum conjugates have been reported. For example, U.S. Pat. No. 5,965,118 describes various platinum chelates attached to the HPMA polymer backbone via a peptide which is potentially cleavable by lysosomal enzymes (see also Gianasi et al. 1999). Additional examples include polyphosphazene platinum (II) conjugates (Sohn et al. 1997; U.S. Pat. No. 5,665,343), poly(glutamate) platinum complexes (Schechter et al. 1987), and others (Bogdanov, Jr., et al., 1996; Han, et al., 1994; Johnsson et al. 1996; Fiebig, et al., 1996); Filipova-Voprsalova et. al., 1991; Fuji et al. 1996; Neuse, et al., 1995; Schechter, et al., 1989).
To our knowledge, none of the above reports of polymer platinum conjugates provides good evidence of structure or nature of the platinum complexation to the polymer, although most make certain unsubstantiated assumptions about the structure of the platinum complex. In all these prior examples, it is possible for platinum to bind to the polymer in more that one way, thus giving rise to the possibility of mixed complexes. Also, pH is not controlled in the formation of the complexes which can lead to the formation of other platinum complexes which an be inactive (hydroxo ligands) or very toxic (aqua ligands). Thus, it is possible that platinum will be released from any one polymer at different release rates, and that the rate of platinum release will vary from batch to batch (as the mixture of complexes formed may vary between batches), giving rise to uncontrolled batch-to-batch variation in both toxicity and efficacy. Such variation is unacceptable for the use of these conjugates in the treatment of cancer. A preferred situation is to have well-defined and well-controlled complexation of platinum to the polymer, that gives a rate of release which is beneficial for the treatment of cancer when utilizing the EPR effect for the improved delivery of platinum compounds to tumors.
In addition to passive tumor targeting utilizing the EPR effect, it may also be possible to target platinum complexes to tumors utilizing xe2x80x98activexe2x80x99 mechanisms. This can be achieved, for example, by the coupling of a platinum complex to a moiety which binds to a receptor which is up-regulated in tumors compared with normal tissue, so giving rise to increased levels of platinum in tumor tissue compared to normal tissue. A wide variety of such up-regulated receptors are known (for example, Heppeler et al, 2000; Schlaeppi et al. 1999; Sudimack et al. 2000; Dubowchik et al. 1999; Weiner, 1999; Buolamwini, 1999). Examples of targeting agents include monoclonal antibodies, peptides, somatostatin analogs, folic acid derivatives, lectins, and polyanionic polysaccharides.
However, to our knowledge there are very few reported examples of the utilization of receptor-targeting mechanisms for the increased delivery of platinum to tumor tissue. Studies of platinum conjugated with monoclonal antibodies (McIntosh et al, 1997; Hata et al, 1992), with steroids (Gust et al, 1995; DiZio et al, 1992, Gibson, et al. 1990) and with folic acid (Vitols et al, 1987), but none have been evaluated in the clinic.
It is also possible to combine the passive targeting of a polymer with the active targeting of a receptor-avid compound. This is exemplified by xe2x80x9cPK2xe2x80x9d, a compound which has a HPMA polymer, doxorubicin attached to the polymer via an enzyme-cleaveable peptide, and is conjugated with galactose, a carbohydrate with strong affinity for the asialoglycoprotein receptor, which is highly concentrated in the liver (Julyan et al, 1999). To our knowledge, this approach, of combining active and passive targeting, has not been explored with platinum chelates.
The present invention is based upon the unexpected discovery of conditions that allow the initial unstable O,O-amidomalonate cis-diamineplatinum(II) complex to rearrange to a pure and isolable N,O-amidomalonate cis-diamine platinum(II) complex. An O,Oxe2x80x94Pt chelate is initially formed when reactive cis-diamineplatinum(II) species react with amidomalonates. Reports discussed below indicate that in such reactions either no N,O-chelate is formed or is found as minor products which were not isolated or purified. Here, general conditions are described which allow a pure N,O-amidomalonate-diamineplatinum(II) to be isolated. Further, such N,O-chelates have preferential biological activity for the treatment of cancer, specifically an improved therapeutic index. The beneficial properties of such N,O-amidomalonate chelates for the treatment of cancer have not previously been reported. Furthermore, the near complete conversion of less thermodynamically-stable complexes to the N,O-chelates described herein provides small molecule and polymeric compounds for treating tumors which can be manufactured with a consistent efficacy and toxicological profile.
For any pharmaceutical product, accurate measures of its identity and purity are necessary. For the present invention and related areas it is most important to verify the exact nature of the platinum complex and identify impurities, for there are examples where an impure platinum complex showed promising biological activity which disappeared upon purification (Talebian et al. 1991 and Appleton et al. 2000).
For the present invention the best method to identify the exact nature of the platinum complex is NMR spectroscopy, specifically 195Pt NMR and 15N NMR spectroscopies (Appleton 2000). With either technique, determination of the identity of platinum complexes is made without the need for prior separation. For this work 195Pt NMR spectroscopy is the method of choice, for it provides sufficient sensitivity and avoids the need for isotopic enrichment required for 15N NMR spectroscopy. 195Pt nuclei are spin xc2xd, possess a receptivity nearly twenty times that of 13C nuclei, and show resonances across a chemical shift range of 15,000 ppm. The chemical shift is very sensitive to the identity and geometry of the platinum ligands. 195Pt has a practical sensitivity limit of about xe2x89xa710 mM platinum. Examples of the chemical shifts for cis-diammine platinum(II) complexes include: xe2x88x922168 ppm for cisplatin, xe2x88x921723 for carboplatin, xe2x88x921584 ppm for diaqua, xe2x88x921841 ppm for monoaqua-monochloro, xe2x88x921732 ppm for O,O-aminomalonate, xe2x88x922156 ppm for N,O-aminomalonate, and xe2x88x922020 ppm for N,O-chelate of N-acetylglycine (Appleton 1990; Gibson 1990; Appleton 2000). Corresponding DACH-Pt complexes appear further upfield.
Reactions of cis-diamineplatinum(II) species with the free amine containing aminomalonate have been documented. Gandolfi (Gandolfi, et al. U.S. Pat. No. 4,614,811; Gandolfi, et al. 1987) reported the preparation and antitumor activity of complexes between cis-diamine platinum(II) species and aminomalonate. The reported structures were all O,O-chelates as shown in FIG. 2a. Later, it was clearly shown (Appleton et al. 1990 and Gibson et al. 1990) that although the O,Oxe2x80x94Pt chelate is formed first, it isomerizes to the thermodynamic N,O-aminomalonate cis-diamineplatinum(II) complex shown in FIG. 2b within a few hours at a pH=5. Furthermore, Appleton showed that if the pH was too low ( less than 2) decarboxylation occurred to give the corresponding N,O-glycine complex. If the pH was too high ( greater than 9) hydrolysis of the platinum ester occurred. Literature reports of the biological activity of pure N,O-aminomalonate complex are not known. However, a report (Talebian 1991) of a closely related well purified N,O-aspartate cis-diamine Pt(II) complex showed little if any cytotoxic activity. For cis-diamineplatinum(II) complexes of amidomalonates like those shown in FIG. 3 (no free amine) Tsujihara in U.S. Pat. No. 4,882,447 reports the preparation and biological activity of a number of O,O-amidomalonate complexes of 1,2-diaminecyclohexaneplatinum(II) (i.e. DACH-platinum(II)). Data verifying the O,Oxe2x80x94Pt chelation was not described. A series of amidomalonate DACH-platinum(II) were reported to only exist as O,Oxe2x80x94Pt chelates like that shown in FIG. 3a (Talebian et al. 1990). A polyphosphazene based amidomalonate was shown as only an O,O-chelate (FIG. 3a), with no confirming spectroscopic data though a similar glutamate based material showed about equal amounts of the two chelates. However, a series of steroid based amidomalonate-diamineplatinum(II) complexes (FIG. 3, R=steroid) were shown to be a mixture of O,Oxe2x80x94Pt and N,Oxe2x80x94Pt amidomalonate chelates (FIGS. 3a and 3b, respectively), with the O,O-isomer being predominant (Gibson et al. 1990). No separation of the two species was described although it was speculated that perhaps with heat or longer reaction times the N,O-chelate could be favored. However, this invention shows that additional components are required to effect the O,Oxe2x80x94Pt to N,Oxe2x80x94Pt conversion of amidomalonate cis-diamine platinum complexes.
In summary, for cis-diamine platinum(II) complexes of aminomalonate the initial O,Oxe2x80x94Pt chelate rapidly isomerizes to the N,Oxe2x80x94Pt chelate. However, for cis-diamine complexes of amidomalonates, either the O,Oxe2x80x94Pt chelate is only found or the O,Oxe2x80x94Pt chelate predominates in mixtures of both chelates. No reports have been found on the preparation of pure N,O-chelate of amidomalonates. Accordingly, the preparation and useful biological activity of the N,O-chelate of cis-diamineplatinum(II) complexes with amidomalonate is now presented. Additionally, the selective preparation of the O,O-chelate is described.
The present invention involves a purified N,O-amidomalonate platinum diamine complex. This complex may be polymer bound. This complex is useful in a method of treating a platinum sensitive neoplasia that involves administering an effective amount of a purified N,O-amidomalonate diamine complex to a patient.
In greater detail, the present invention involves a composition for use in tumor treatment, comprising a cis-diamine N,O-amidomalonate platinum species of the form: 
where R1 is H, alkyl, a water solubilizing group, carrier or a targeting group useful for targeting the species to a tumor; R2 and R3 are amines; R4 is H or a cation; and where said species has, or is converted in vivo to have, anti-tumor activity. The cation in this complex may be an ammonium ion, an alkali, or an alkali earth metal. A preferred cation is sodium.
In certain cases, the N,O-amidomalonate platinum diamine complex may involve the above composition, wherein R1 is a synthetic polymer of N-alkyl methacrylamide units of molecular weight from 1-5000 kDaltons and the form: 
where m=0 and n=100 or where the ratio of m:n is 0.1-99.9; where R5 is H or CH3; where R6 is a C1-C6 hydroxyalkyl group and where R7 is an oligopeptide chain capable of being cleaved under physiological conditions with the sequence of Gly-(W)P-Gly where p is 0-3 and W is an amino acid or combination of any amino acids and whose C terminus is an amide of the amido malonato group.
In an important embodiment of the above N,O-amidomalonate platinum complex both R2 and R3 are in NH3. These are often preferably, the primary amine groups of 1,2-diaminocyclohexane.
The platinum involved in these complexes may be in the +2 or +4 oxidation state. R1 as mentioned above is either H or alkyl, but may also be a steroid or a folic acid or a folic acid derivative or analog useful to target folate receptors.
The polymer of the polymer N,O-amidomalonate platinum complex may be, along with other polymers described herein, a polyglutamic acid, a mono- or polysaccharide or the side chain of a polysaccharide.
The present invention also involves a method of improving the stability of a platinum diamine compounds. This method involves forming a purified N,O-amidomalonate complex of the platinum compound.
In an important aspect of the present invention involves a composition for use in tumor treatment, comprising a polymer-platinum complex designed to accumulate at a tumor site and composed of an N-alkyl acrylamide polymer having side chains spaced along the polymer for complexing with a platinum compound, said side chains (i) composed of an oligopeptide attached at one end to the polymer and at the other end, at least primarily via a N,O-amidomalonate complex, to the platinum compound and (ii) including at least one linkage designed to be cleaved under selected physiological conditions to yield a platinum compound which has, or is converted in vivo to have, anti-tumor activity.
Such an N-alkyl acrylamide polymer is preferably a homopolymer having a molecular weight of between about 1,000 and about 5,000,000 Daltons. The N-alkyl acrylamide polymer may be a copolymer having two repeat units m and n in a ratio m:n of between 0.1 and about 99.9.
The compositions of the present invention may also involve repeat units of an N-alkyl acrylamide unit carrying oligopeptide side chains. These oligopeptide side chains may terminate in a proximal group capable of attaching the platinum compound.
In the compositions of the present invention, the useable polymer may be a copolymer of the form where the polymer is a copolymer of the form: 
where R1 is H or CH3, R2 is a lower alkyl or lower hydroxyalkyl group, and R3 is an oligopeptide side chain. In this polymer, R1 is CH3, R2, is 2-hydroxypropyl, and R3 is Gly-Phe-Leu-Gly-Ama or Gly-Gly-Ama. In therapeutic uses of the present invention, the polymer platinum compound is dissolved in a aqueous medium suitable for parenteral administration.
An important aspect of the present invention is a method of treating a solid tumor in a subject with a platinum compound, the method comprising preparing a polymer-platinum complex composed of an N-alkyl acrylamide polymer having side chains spaced along the polymer for complexing with a platinum compound, said side chains (i) composed of an oligopeptide attached at one end to the polymer and at the other end to the platinum compound via a N,O-amidomalonate complex and (ii) including at least one linkage designed to be cleaved under selected physiological conditions to yield the platinum compound which has, or is converted in vivo to have, anti-tumor activity; and parenterally administering a pharmaceutically effective amount of the complex to the subject. Said N-alkyl acrylamide polymer in one preferable embodiment is a homopolymer having a molecular weight of between about 1,000 and about 5,000,000 Daltons. In another important embodiment the N-alkyl acrylamide polymer is a copolymer having a molecular weight between 1,000 and 5,000,000 Daltons. This copolymer contains two repeat units m and n in a ratio of m:n between 0.1 and about 99.9. Such repeat units comprise an N-alkyl acrylamide unit and a unit carrying an oligopeptide side chain having a proximal end capable of attaching to a platinum compound. When an oligopeptide is used, said oligopeptide is preferably Gly-(W)p-Gly where p is 0-3 and W is an amino acid or combination of any amino acids. In one important embodiment the oligopeptide is Gly-Phe-Leu or Gly-Gly.
This invention comprises a method enhancing the therapeutic index of a platinum diamine compound when the compound is used for treating a tumor by parenterally administering a pharmaceutically acceptable solution containing the compound to a subject, comprising prior to said administering, complexing the platinum compound with a copolymer composed of an N-alkyl acrylamide first repeat unit and a second repeat unit having an oligopeptide side chain having an amidomalonate end group complexing via N,O linkages with said platinum compound.
From another view, this invention involves a method of improving the stability of a platinum diamine compound comprising complexing the compound with a copolymer composed of an N-alkyl acrylamide first repeat unit and a second repeat unit having an oligopeptide side chain having an amidomalonate end group complexing with said platinum compound through an O,N-linkage.
Accordingly, it is an object of the invention to provide new polymer-platinum complexes having improved antitumor activity in vivo.
In one aspect, the invention includes a composition for use in tumor treatment, comprising polymer-platinum compounds designed to accumulate at a tumor site. The compound is composed of a synthetic polymer backbone having platinum-containing side chains attached to the backbone. The side chains (i) are composed of a biodegradable linker, for example, an oligopeptide attached at or near one end to the backbone and at or near the other end to a platinum compound. The linker includes at least one linkage which is designed to be cleaved under selected physiological conditions to yield the platinum compound which has, or is converted in vivo to have, anti-tumor activity. The oligopeptide may contain more than the usual amino acids, e.g., aminomalonate and the like or other than alpha amino acids.
In one embodiment, the synthetic polymer is a homopolymer of an N-alkyl acrylamide or methacrylamide (i.e. all xe2x80x98nxe2x80x99 type repeat units) having a molecular weight of between about 1,000-5,000,000 Daltons.
In another embodiment, the synthetic polymer is a copolymer having a molecular weight between 1,000 and 5,000,000 Daltons and contains two repeat units m and n in a ratio m:n of between about 0.1 and 99.9.
The repeat units, in one embodiment, are composed of an N-alkyl acrylamide or methacrylamide unit and of a unit carrying the oligopeptide side chain which terminates in a proximal end group capable of attaching the platinum compound.
In one embodiment, the polymer in the polymer-platinum compound is a copolymer of the form: 
where m=0 and n=100 or where the ratio of m:n is 0.1-99.9;
where R1 is H or CH3, R2 is a lower alkyl or lower hydroxyalkyl group, and R3 is a oligopeptide side chain.
The oligopeptide is, in another embodiment, an oligopeptide of the form Gly-(W)p-Gly where p can be 0 to 3 and (W) can be any amino acid or combination of any amino acid. In one embodiment, the peptide is Gly-Phe-Leu-Gly and terminates in a carboxyl, diamine or malonyl moiety for attachment to the platinum compound. The Phe or Leu are (L) amino acids in the preferred embodiment. In another embodiment, the peptide is Gly-Gly terminating in a proximal carboxyl end group. To the extent that D-amino acid-containing oligopeptides are biodegradable, they too may be part or all of an oligopeptide.
In a preferred embodiment, R1 is CH3, R2 is 2-hydroxypropyl, and R3 is Gly-Phe-Leu-Gly-[X] where [X] is a diamine, a carboxyl group or a malonyl moiety.
The polymer-platinum compound is dissolved in a pharmaceutically acceptable medium suitable for parenteral administration.
In another aspect, the invention includes a method of targeting a platinum compound to a solid tumor in a subject. The method includes preparing a polymer-platinum compound composed of a synthetic polymer backbone having side chains spaced along the backbone. The side chains (i) are composed of an oligopeptide attached at one end to the backbone and at the other end to a platinum compound and (ii) include at least one linkage which is designed to be cleaved under selected physiological conditions to yield the platinum compound which has, or is converted in vivo to have, anti-tumor activity. The compound is parenterally administered in a pharmaceutically effective amount to the subject.
In another aspect, the invention includes a method of enhancing the therapeutic index of a platinum compound, when the compound is used for treating a tumor by administering parenterally a pharmaceutically acceptable solution containing the compound to a subject. The method includes, prior to administering the compound, complexing the platinum compound with a copolymer composed of an N-alkyl acrylamide first repeat unit and a second repeat unit having an oligopeptide side chain which terminates in a proximal end group capable of complexing with the platinum compound.
In another aspect, the invention includes a method of improving the solubility and/or stability of a platinum compound by complexing the compound with a copolymer composed of an N-alkyl acrylamide first repeat unit and a second repeat unit having an oligopeptide side chain which terminates in a proximal end group capable of complexing with said platinum compound. The polymer-platinum complex is more soluble and/or more stable under physiological conditions than non-complex platinum compounds. A preferred platinum complex is bound throughxe2x80x94and O- of most preferably an amidomalonate residue connected to a biodegradable linkage to a polymer.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.
Polymer-based delivery of therapeutic agents, including chemotherapeutic drugs, continues to receive considerable attention (Duncan et al (1999), Seymour). Typically, a well-established pharmacological entity is chemically linked to a biologically inert polymer, thus profoundly altering its distribution, elimination, and toxicological properties. For oncological applications, this technology offers the potential of increasing the concentration of the cytotoxic agent within the tumor interstitium via the enhanced permeability and retention (EPR) effect (Seymour, et al). ACCESS Pharmaceuticals has rights to a broad class of platinated polymer therapeutics. One of these, designated AP 5280, is a 90:10 copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and the methacrylamide of Gly-Phe-Leu-Gly with an aminomalonato chelate of cis-diammineplatinum(II). Incorporation of this optimized linker offers the potential to release platinum-containing fragments from the polymer via cleavage by tumor proteases. The concept of this copolymer-linker-chelate combination, and early synthetic and biological studies, have been presented by Duncan et al (1999). The challenge in further developing this material for clinical evaluation has been to define a scalable procedure for a structurally-characterized product having the requisite activity, stability, and pharmaceutic properties necessary to secure regulatory approval for use in humans.
The synthesis of AP 5280 is accomplished by initially substituting diethyl aminomalonate for p-nitrophenol in the intermediate poly(HPMA) GFLG-ONp to give poly(HPMA)-GFLG-Ama-diEt. The latter is saponified, then platinated with cis-(NH3)2Pt(H2O)22+ to give poly(HPMA)-GFLG-Ama=Pt(NH3)2. This is followed by the controlled rearrangement of the initial O,Oxe2x80x94Pt chelate to the N,Oxe2x80x94Pt chelate. We have also made (Polymer Labs) the poly(HPMA)-GFLG-Ama-diEt from the HPMA and MA-GFLG-Ama-diEt monomers. By polymerizing these monomers with various amounts of a radical chain transfer agent (i.e. p-nitrophenol) the molecular weight is controlled. (Note: this method of control of molecular weight is well known in the literature.) These polymers were then saponified, platinated, and rearranged to give the N,O-chelate as described. Purification from low-molecular weight impurities is achieved by tangential-flow filtration, with isolation of the final formulated product by terminal lyophilization. The identity and purity of the N,Oxe2x80x94Pt chelate ( greater than 92%) is confirmed by 195Pt NMR spectroscopy (xe2x88x922056 ppm), with  less than 8% Pt present as the O,O-chelate (xe2x88x921733 ppm) or other Pt species. The final product contains 8.0xc2x10.5% Pt (wt/wt) and has a Mw=24.4 kDa.
In water, AP 5280 releases  less than  less than 1% of the platinum content as polymer-free platinum species, and releases  less than 2% of the platinum into medium containing physiological concentration of chloride over 24 hours at 37xc2x0 C. The efficacy of AP 5280 was evaluated in a s.c. B16F10 murine tumor model, which showed activity at 20 mg Pt/kg equivalent to that of cisplatin at 3 mg/kg. Activity superior to carboplatin (45 or 60 mg/kg) is achieved with AP 5280 at 200 mg Pt/kg (all doses IV, qdxc3x975). In an important aspect, Figure A summarizes the process for the AP 5280 procedure.